Assay for monitoring activity of frizzled receptors

ABSTRACT

The present invention relates to cell free assays for measuring receptor activity, especially for measuring a constitutive or a non-constitutive activity of frizzled receptors and uses thereof. The present invention further concerns a method for measuring a constitutive or non-constitutive activity of a frizzled receptor and a method for obtaining an active frizzled receptor ligand.

FIELD OF THE INVENTION

The present invention relates to the technical field of cell free assaysfor measuring receptor activity, especially for measuring a constitutiveor non-constitutive activity of frizzled receptors. The presentinvention further concerns a method for measuring a constitutive ornon-constitutive activity of a frizzled receptor and a method forobtaining an active frizzled receptor ligand.

BACKGROUND OF THE INVENTION 1. Cell Signaling

Cells use a large number of clearly defined signaling pathways toregulate their activity. These signaling pathways fall into two maingroups depending on how they are activated. Most of them are activatedby external stimuli and function to transfer information from the cellsurface to internal effector systems. However, some of the signalingsystems respond to information generated from within the cell, usuallyin the form of metabolic messengers. For all of these signalingpathways, information is conveyed either by protein-protein interactionsor it is transmitted by diffusible elements usually referred to assecond messengers or transmitters. The ability of cells to perceive andcorrectly respond to other cells is the basis of development, tissuerepair, and immunity as well as normal tissue homeostasis. Errors incellular information processing are responsible for diseases such ascancer, autoimmunity, and diabetes.

2. Wnt Proteins and Frizzled Receptors

Secreted Wnt (Wg Int) proteins have numerous signaling functions duringdevelopment, mediated by certain receptors on the cell surface. Theseproteins are defined by their sequence rather than by functionalproperties. They contain a signal sequence of 350-380 amino acidsfollowed by a highly conserved distribution of cysteines. Although Wntproteins are secreted they show an insoluble nature that has beenexplained by the discovery that these proteins are palmitoylated and aremore hydrophobic than initially predicted from the primary amino acidsequence. The palmitoylation is found on a conserved cysteine,suggesting that all Wnts essentially carry this modification (Willert K.et al., 2003). Until now this insoluble nature of all members of the Wntfamily has hindered attempts to purify the Wnts and precluded anisolation of Wnts in high quantities.

Wnts proteins play diverse and essential roles in generation of cellpolarity, embryonic induction, specification of cell fate, and diseasessuch as cancer or degeneration. At the molecular level, Wnt proteinsoperate largely via receptor-mediated signaling pathways, and thesereceptors appear to be members of the frizzled family (Huang H. andKlein P., 2004).

Frizzled receptors (Fz) are integral membrane proteins withtransmembrane domains, an exposed binding site outside the cell and aneffector site extending into the cytosol. They function in multiplesignal transduction pathways and have been identified in numerousanimals, from sponges to humans. The family is defined by conservedstructural features, including seven hydrophobic transmembrane domainsand a cysteine rich ligand-binding domain (Huang H. and Klein P., 2004).

Fz function in three distinct signaling pathways, known as the planarcell polarity pathway, the canonical Wnt/β-catenin pathway, and theWnt/calcium pathway. The cytoplasmic Fz domains that link toheterotrimeric G proteins and other downstream signaling componentstransduce a signal to several intracellular proteins that includedishevelled, glycogen synthase kinase-3β (GSK-3β), axin, adenomatouspolyposis coli (APC), and the transcriptional regulator β-catenin.Cytoplasmic β-catenin levels are normally kept low by continuousproteasome-mediated degradation, which is controlled by a complexcontaining GSK-3/APC/Axin. When cells receive Wnt signals, thedegradation pathway is inhibited, and consequently β-catenin accumulatesin the cytoplasm and nucleus. Nuclear β-catenin interacts withtranscription factors such as lymphoid enhancer-binding factor 1/Tcell-specific transcription factor to affect transcription. A largenumber of Wnt targets have been identified that include members of theWnt signal transduction pathway itself, which provide feedback controlduring Wnt signaling (Logan C. and Nusse R., 2004).

Secreted proteins of the Wnt family play widespread roles in theregulation of embryonic development, and aberrant activation of thecanonical Wnt/β-catenin pathway is one of the most frequent signalingabnormalities known in human cancer. In human breast cancer, evidence ofβ-catenin accumulation implies that the canonical Wnt signaling pathwayis active in over 50% of carcinomas (Brennan K. and Brown M., 2004).

Much of last years' research focused on the development of cell basedassays and screening techniques for drug discovery and design. Screeningof cell signaling pathways in primary cells of a physiologicallyrelevant phenotype provides a means to identify modulators of importantdisease pathways that lack known drug targets.

Borchert K. et al. (2006) screened for small molecule activators of Fzin primary human preosteoblasts that should contain intact andphysiologically relevant Wnt/FZ signaling components. For this purposethey measured endogenous translocation of the downstream transcriptionfactor β-catenin to the nucleus by combining standard immunofluorescenttechniques with automated fluorescence microscopy.

DasGupta R. et al. (2005) used the availability of the Drosophila genomesequence, to find new components in the Wnt signaling pathway. By RNAinterference (RNAi) based screening technology they identifiedfunctional genes regulating the Wnt-Wg pathway. The assay for the RNAiscreen was based on the Wnt reporter TOP-Flash (TCF optimal promoter),which consists of multimerized TCF-binding sites driving the expressionof a cDNA encoding the firefly luciferase gene. The screen was performedin Drosophila imaginal disc-derived clone 8 cells, which are epithelialin origin. The activity of the Wg signaling pathway was quantified bymeasurement of normalized luciferase expression or relative luciferaseactivity units, which equated to the ratio of the absolute activity offirefly luciferase to that of renilla luciferase.

Signal transduction has been studied extensively with cell basedsystems, but interest and commercial investment in Fz in areas such asdrug targets, orphan receptors, high throughput screening, andbiosensors, among others will focus greater attention on cell free assaydevelopment to allow for miniaturization, ultrahigh throughput and,eventually, microarray/biochip assay formats. Although cell based assaysare adequate for many Fz, these formats would limit the development ofhigher density Fz assay platforms mandatory for other applications.Stable, robust and cell free signaling assemblies comprising receptorand appropriate molecular switching components form the basis of futureFz assay platforms adaptable for such applications as microarrays. Inaddition cell free assays ensure a uniform response resulting inwell-defined mechanisms of action and no ambiguity of experimental datadue to other interfering and interactive pathways within the cell.Another advantage of cell free assays is the free accessibility of thecompound to the target.

Thus there is a need for a fast and convenient cell free assay tomeasure the activity of Fz that is suitable for high throughputscreening.

The solution to this problem is achieved by providing the embodimentscharacterized by the claims, and described further below.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a cell free assayfor measuring a constitutive activity of frizzled receptors comprisingat least one G protein, at least one system for measuring G proteinactivity and at least one membrane protein, wherein a concentration ofthe G protein is approximately 0.4 μg/ml or less, preferably betweenapproximately 0.002 μg/ml and 0.3 μg/ml, more preferably betweenapproximately 0.005 μg/ml and 0.15 μg/ml, most preferably approximately0.1 μg/ml in relation to approximately 2 μg/ml to 15 μg/ml of themembrane protein.

Furthermore the invention concerns a cell free assay for measuring anon-constitutive activity of frizzled receptors comprising a G protein,at least one system for measuring G protein activity and at least onemembrane protein, wherein a concentration of the G protein isapproximately 0.3 μg/ml or higher, preferably between approximately0.035 μg/ml and 10 μg/ml, more preferably approximately 0.4 μg/ml, mostpreferably approximately 0.5 μg/ml in relation to approximately 2 μg/mlto 15 μg/ml of the membrane protein.

In addition, the present invention is directed to a method for measuringa constitutive or non-constitutive activity of a frizzled receptorcomprising the steps of:

-   -   a) providing at least one membrane protein with at least one        cell free frizzled receptor;    -   b) adding at least one frizzled receptor ligand and at least one        G protein; and    -   c) incubating the cell free frizzled receptor, the frizzled        receptor ligand and the G protein in a system for measuring G        protein activity, wherein the detection of guanosine        triphosphate (GTP) binding to the G protein indicates the        activity of the G protein.

The present invention is also directed to method for obtaining an activefrizzled receptor ligand comprising the steps of:

-   -   a) performing random and/or directed mutagenesis in at least one        polynucleotide sequence encoding at least one inactive frizzled        receptor ligand to generate a mutated polynucleotide sequence;    -   b) digesting the mutated polynucleotide sequence into random        polynucleotide fragments;    -   c) recombining the random polynucleotide fragments to obtain        recombinant polynucleotides by at least one recombination        technique;    -   d) cloning the recombinant polynucleotide into a vector;    -   e) expressing the recombinant polynucleotide resulting in        recombined frizzled receptor ligands;    -   f) measuring the ability of the recombinant frizzled receptor        ligands to activate frizzled receptors in a system comprising at        least one G protein and at least one membrane protein with at        least one frizzled receptor; and    -   g) repeating step a) to f) until the recombinant frizzled        receptor ligands activate the G protein at least 10-fold,        preferably 100-fold, more preferably 500-fold, most preferably        1000-fold compared to frizzled receptor ligands encoded by the        polynucleotide sequence of step a).

Furthermore the invention relates to the use of an assay for screeningfrizzled receptor ligands and/or for measuring levels of frizzledreceptor ligands and/or for measuring levels of frizzled receptorsand/or for obtaining active frizzled receptor ligands, wherein the assaycomprises at least one G protein and at least one system for measuring Gprotein activity. A concentration of the G protein is approximately 0.4μg/ml or less, preferably between approximately 0.002 and 0.3 μg/ml,more preferably between approximately 0.005 and 0.15 μg/ml, mostpreferably approximately 0.1 μg/ml in relation to approximately 2 to 15μg/ml of membrane protein for measuring constitutive G protein activity,or is approximately 0.3 μg/ml or higher, preferably betweenapproximately 0.035 and 10 μg/ml, more preferably approximately 0.4μg/ml, most preferably approximately 0.5 μg/ml in relation toapproximately 2 to 15 μg/ml of membrane protein for measuringnon-constitutive G protein activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A) shows membrane fractions of control bacteria and twobacterial clones expressing a maltose binding protein (MBP) humanFrizzled receptor 1 (hFz1) fusion protein (MBP-hFz1) in a western blotwith anti-MBP.

FIG. 1 (B) shows membrane fractions of control bacteria and twobacterial clones expressing hFz1 in a western blot with anti-Gαo.

FIG. 2 shows the correlation of the constitutive G protein activity ofhFz1 to the concentration of added G protein Go.

FIG. 3 (A) hFz1 membranes generated by CHO were activated by Wnt3a, E.coli-produced hFZ1 membranes were activated by purified Wnt3a, and forcomparison, CHO membranes expressing human fMLP receptor hFRP1 wereactivated to a similar extent by a formyl-peptide ligand.

FIG. 3 (B) produced hFz1 membranes generated by E. coli were stimulatedwith a Wnt5a-mimetic peptide.

FIG. 4 shows GTP incorporation into Go in the presence of different Wntligands. Incorporation is stimulated by detergent-solubilized hFz1.

FIGS. 5 (A)-(C) show stimulation of several Frizzled receptors by Wntligands to activate the trimeric G proteins.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a cell free assay for measuring aconstitutive activity of frizzled receptors comprising at least one Gprotein, at least one system for measuring G protein activity and atleast one membrane protein, wherein a concentration of the G protein isapproximately 0.4 μg/ml or less, preferably between approximately 0.002μg/ml and 0.3 μg/ml, more preferably between approximately 0.005 μg/mland 0.15 μg/ml, most preferably approximately 0.1 μg/ml in relation toapproximately 2 μg/ml to 15 μg/ml of the membrane protein.

The term “cell free assay” as used herein refers to an assay, wherein noentire or intact cells are used. Cells can be disrupted by methods likeenzymatic dissociation by lysozym; physical dissociation by ultrasound,French press, or glass-rod homogenizer; or non-isotonic buffers ashypotonic buffer with 10 mM Hepes etc. Disrupted cells exist as membranefractions or cells with membranes perforated by antibiotics likegramicidine. The term “cell free” also means that receptors of the cellmembranes can be coupled to a solid phase. Cell free assays areadvantageous, because they are very robust and well suited for highthroughput methods in micro formats. Their results are uniform and donot interfere with cellular interactions. Besides, cell freepreparations provide a free accessibility to molecules of interest.

The term “constitutive activity” as used herein refers to a basalactivity or intrinsic activity of receptors or proteins that does notdepend on the presence of an exciting ligand or agonist of the receptor.Constitutive active receptors and proteins can spontaneously form activestates that subsequently can activate downstream molecules like Gproteins, thus producing a measurable response.

The term “frizzled receptors” as used herein refers to a family ofheptahelicale serpentine receptors with integral membrane proteinscomprising transmembrane domains, an exposed binding site outside thecell and an effector site extending into the cytosol. They range inlength from about 500 to 700 amino acids forming a low densitylipoprotein (LDL) receptor-related protein (LRP) complex at the cellsurface. The amino terminus is predicted to be extracellular andcontains a cysteine rich domain followed by a hydrophilic linker regionof 40-100 amino acids. The proteins also contain seven hydrophobicdomains that are predicted to form transmembrane helices. Theintracellular carboxyterminal domain has a variable length and is notwell conserved among different family members. A motif located two aminoacids after the seventh hydrophobic domain is highly conserved in fzgenes and is essential for activation of the Wnt/catenin pathway. Alsocomprised are frizzled-related proteins known as Frzb, that arehomologous to Fz expressed e.g. in chondrocytes.

The fz genes were first identified in Drosophila and subsequently foundin diverse metazoans, in vertebrates as fz1-10 and in Caenorhabditiselegans. Several fz genes appear to lack introns, however, includingvertebrate orthologs of human fz1, fz2, and fz7 to fz10. Other fz genes,such as human fz5 and Drosophila frizzled 2, contain one intron, but theentire open reading frame is encoded by a single exon.

Fz are found exclusively at the plasma membrane. They are located at thesurface of Wnt-responsive cells, but may be internalized as part of amechanism for regulating the extracellular level of Wnt protein and/orthe cellular response to Wnt proteins.

Fz are typically coupled to trimeric G protein complexes. A G proteincomplex consists of a GDP bound α subunit and a βγ dimer and associateswith intracellular portions of receptors. The term “activity of frizzledreceptors” as used herein refers to activated receptors that catalyzeexchange of GDP for GTP on Gα subunit. Dissociation of the complexesfollows and the released Gα-GTP and βγ moieties are then free to engagewith downstream effectors. By the time, the α subunit hydrolyzes GTP toGDP, generating the reformation of the complex and reassociation withthe receptor.

Fz function in three distinct signaling pathways, known as the planarcell polarity (PCP) pathway, the canonical Wnt/β-catenin pathway, andthe Wnt/calcium pathway as mentioned above. The PCP pathway is definedby the set of genes that, when mutated, result in defects in thepolarity of cells in a planar tissue. Studies in mutant zebra fish andXenopus suggest a role for Fz in oriented gastrulation movements invertebrate, in which a convergent-extension phenotype arise throughdisruption of a PCP pathway. Asymmetric subcellular distribution of Fzhas a central role in establishing cell polarity in flies and mostlikely in other organisms as well.

The canonical Wnt/β-catenin pathway is characterized by stabilization ofβ-catenin protein in response to ligand binding. β-catenin proteinconnects actin filaments to cadherins to build up adherent junctionsbetween cells. Any excess of β-catenin is quickly destroyed by amultiprotein degradation complex that is partially encoded by tumorsuppressor genes, e.g. APC tumor suppressor gene. In the absence of Wnt,GSK3 acts unopposed to phosphorylate both the scaffold axin as well asthe β-catenin, which destabilizes β-catenin, directing it to degradationby the proteosome. In the presence of Wnt, Fz1 action inactivates GSK3β,stabilizes β-catenin, and leads to accumulation and translocation ofβ-catenin to the nucleus, where it combines with members of the Tcffamily of DNA-binding proteins, enabling transcription. β-catenin hasbeen shown to compete with the transcriptional repressor Groucho,transforming the transcription factor Tcf from a repressor to anactivator (Huang H. and Klein P., 2004).

The term “G protein” as used herein refers to G proteins, short forguanine nucleotide binding proteins that are a family of proteinsinvolved in second messenger cascades. They are called “G proteins”because of their signaling mechanisms, which use the exchange ofguanosine diphosphate (GDP) for guanosine triphosphate (GTP) as ageneral molecular switch function to regulate cell processes. G proteinsbelong to the large group of GTPases. “G protein” usually refers to themembrane-associated heterotrimeric G proteins, sometimes referred to asthe large G proteins. These proteins are activated by G protein-coupledreceptors and are made up of alpha (α), beta (β) and gamma (γ) subunits.The definition for “G protein” used herein also includes small Gproteins that belong to the Ras super-family of small GTPases. Theseproteins are homologous to the α-subunit found in heterotrimers and arein fact monomeric. However, they also bind GTP and GDP and are involvedin signal transduction. G proteins are perhaps the most important signaltransducing molecules in cells. In fact, diseases such as cancer,diabetes, and certain forms of pituitary cancer, among many others, arethought to have some root in the malfunction of G proteins.

The term “G protein activity” as used herein refers to a G protein in anactive state, wherein it is not bound to GDP. GDP bound G protein isinactive. Typically the inactive G protein is bound to its receptor.Once the ligand is recognized, the receptor, e.g. Fz, shifts itsconformation and thus mechanically activates the G protein, whichdetaches from the Fz. In addition, the G protein activity can be a basalor constitutive activity that is spontaneous and not ligand gated. It isbelieved that G proteins and Fz exist in a conformational equilibriumbetween active and inactive biophysical states. The binding of ligandsto Fz may shift the equilibrium toward the active Fz states. Accordingto the definition used herein activated G proteins are bound to GTPdisregarding whether G proteins are bound or not bound to thecorresponding receptor. Also included are partially activated anddesensitized G proteins. Activation of G protein catalyzes the exchangeof GDP for GTP on Gα subunit, the G protein complex gets instable andtends to dissociate. By the released Gα-GTP and βγ moieties activated Gproteins engage downstream effectors.

The term “system for measuring G protein activity” as used herein refersto a configuration, wherein the amount of active G protein is measuredin a direct or indirect way. Therefore the amount of active or inactiveG protein can be measured as well as transitions of an inactive to anactive state and vice versa. The active or inactive state or thetransitions between these states of downstream or upstream molecules ofG proteins can also be quantified. In addition, the configuration canquantify the amount of bound and unbound ligands of G proteins; or theamount of down- or upstream molecules; or binding and release of theseligands. In a preferred embodiment the interaction between G protein andGDP or GTP, respectively, is detected. In the most preferred embodimentthe GTP binding to G protein is detected by using labelled GTP.

The term “membrane protein” as used herein refers to all membraneproteins that are included in the phospholipid double or mono layer ofcells, vesicles, compartments, membrane fragments, reconstituteddetergent-containing or micelle-containing solutions or suspensions. Fzare a component of membrane proteins. Apart from Fz these membraneproteins may contain other membrane associated proteins. Thequantitative portion of Fz in the overall amount of membrane proteins(Fz/total) depends on the design of expression system and experimentalpreparation of the membrane protein. The indicated values of the presentinvention concerning the concentration ratios of G protein to membraneprotein are based on expression systems in bacteria and Chinese hamsterovary cells (CHO), and cell free preparations of membrane fractions asherein described in Materials and Methods, chapter 1 and 2. Otherprotein expression systems and membrane protein preparations may resultin different Fz/total ratios. For example, in systems overexpressing FzFz/total is increased, whereas in systems with poor transfectionefficiency Fz/total is decreased. Due to these possible variations ofFz/total the indicated values of the present invention concerning theconcentration ratios of G protein to membrane protein have to beadjusted if expression and/or preparation conditions result in Fz/totalthat deviates compared to the Fz/total of the present invention. Thisadjustment has to put into effect that the ratio of G protein to Fz isaccording to the present invention. For instance, if the Fz/total ofother systems is higher compared to the invention by e.g. Fzoverexpression, the concentration ratio of G protein to membrane proteinof the invention has to be increased, whereas if the Fz/total of othersystems is lower compared to the invention by e.g. poor transfectionefficiency, the ratio of G protein to membrane protein of the inventionhas to be decreased.

In a preferred embodiment of the invention membrane proteins are Fz.They can be provided in any kind of preparation e.g. in crudepreparations, as extracts, purified, cell free as membrane fractionsetc.

The present invention is based on the new and important finding that Fzare constitutively active receptors. As can be seen from FIG. 2, humanfrizzled receptors exemplified by hFz1 possess a strong constitutiveactivity towards trimeric G proteins. A very surprising effect was thatthis constitutive activity is restricted to a certain range of G proteinconcentrations. Typically one would expect that the higher the G proteinconcentration is the more constitutively active the Fz are until asaturated status with a constant activity is achieved. In contrast tothis, higher G protein concentrations i.e. higher Go/Fz ratios led to aninhibition of the constitutive activity of G protein or Fz,respectively. This unexpected phenomenon is demonstrated by a bellshaped concentration dependent curve of the constitutive activity ofhFz1.

Without intending to be bound by any theory it is believed that thisconcentration dependency of constitutive Fz activity can be explained bya negative feedback regulation. A constitutive active Fz activates Go,producing Gαo-GTP, and Gαo-GTP in turn inhibits Fz from generating moreGαo-GTP. Therefore, Gαo-GTP can inhibit its own production by inhibitingFz to catalyze GDP/GTP exchange on the trimeric Go complexes. Thus,concentrations of Gαo-GTP and consequently constitutive G proteinactivity are well balanced.

Such a negative feedback interaction is unprecedented for receptorstransducing via G proteins. Biologically, it might have the significanceof ensuring a very transient activation of the intracellular signaltransduction upon ligand addition to Fz. In fact, in the case of Fzcontrolled planar cell polarity signaling in Drosophila, mathematicalmodelling has predicted a very short time scale of Fz activation as aprime requirement for the proper planar cell polarity establishment (LeGarrec J. et al., 2006). This mathematical modelling is interpreted by ashort-lived ligand that activates Fz. In contrary to this interpretationof Le Garrec J. et al., the data herein concerning the inhibition ofconstitutive activity with increasing G protein concentration suggest anegative feed-back in the Fz-Go interactions. Therefore, the Fz liganddoes not have to be short-lived, but the intrinsic property of the Fz-Gointeraction insures a short time period for active Fz by the negativefeed-back.

On the basis of the constitutive activity of Fz or Fz coupled G proteinsthat is restricted to a certain range G protein concentration thepresent inventors developed a new assay for measuring the constitutiveactivity of Fz. In the assay of the invention the concentrations of Gprotein to Fz are provided in ratios, wherein the Fz or the Fz coupled Gproteins are constitutively active. The assay makes use of this welldefined restricted concentration range, wherein Fz or Fz coupled Gproteins are specifically constitutively active. By experimentallypresetting the concentration ratio of G protein/Fz to low values theassay defines the kind of Fz activity that can be modulated by addingligands, modulators, effectors etc. Therefore, the present inventionprovides an assay for measuring specifically the constitutive activityof Fz.

By the assay of the present invention for measuring the constitutive Fzor G protein activity the influence of Fz ligands, e.g. Fz agonists, Fzantagonists, Fz modulators, Fz effectors etc., as well as all kind ofmodulators that are associated with the Fz pathway, e.g. effectors thatbind apart from Fz e.g. to G proteins, inverse agonists, which switchoff or reduce constitutive activity can be determined. It can beenvisioned that e.g. an inverse agonist of the constitutive Fz activitywill have an application as a drug reducing Fz activity in e.g. breastcancer or dementia. Therefore, this assay provides a high throughputscreening technique to find pharmaceutical modulators of Fz activitiesin a simple, robust and fast manner.

The assay of the present invention for measuring the constitutive Fz orG protein activity is extremely useful for investigating Fz/ligandinteractions and for screening Fz ligands. If optimal concentrations ofGo for the constitutive hFz1 activity are used, Fz can not be activatedfurther by ligand binding, but instead a 20%±10% reduction in Fzactivity is produced upon binding of e.g. an agonists.

Another advantage of this assay of the present invention is itsutilisation for orphan Fz, whose function and/or ligands are stillunknown. Measuring the constitutive activity of these orphan receptorsby the assay of the present invention is the only way to investigate theinteraction of modulators, effector and ligands with Fz orphanreceptors. Therefore the assay is ideal for discovering and screeningunknown orphan receptor ligands producing a measurable response.

Another favorable property of the assay of the present invention formeasuring the constitutive Fz or G protein activity is the fact that invitro binding of Go to Fz as showed in FIG. 1 is not inhibited byaddition of GTP or GTPγS. These and other data of the present inventorreveal the unexpected property of the Go-Fz interactions, that theseinteractions persist even when the trimeric Go complex is dissociatedinto the GTP-loaded Gαo subunit and the beta-gamma heterodimer. Suchpersistence is undisclosed so far in the prior art and is believed to benot the case for the majority of other GPCRs and their cognate Gproteins. Therefore, the measurements by the assay of the presentinvention concerning the constitutive Fz or G protein activity are notdistorted by varying GTP concentrations.

The present invention is also directed to a cell free assay formeasuring a non-constitutive activity of frizzled receptors comprising aG protein, at least one system for measuring G protein activity and atleast one membrane protein, wherein a concentration of the G protein isapproximately 0.3 μg/ml or higher, preferably between approximately0.035 μg/ml and 10 μg/ml, more preferably approximately 0.4 μg/ml, mostpreferably approximately 0.5 μg/ml in relation to approximately 2 μg/mlto 15 μg/ml of the membrane protein.

The term “non-constitutive activity” as used herein refers to anactivity of a receptor or protein that is dependent on the presence ofan exciting ligand or an agonist that binds to the receptor causing aconformational change that is transmitted for activating downstreammolecules. The non-constitutive activity is also called transmitterinduced activity.

The present invention is based on the finding that Fz can be activatedin a non-constitutively way. As can be seen from FIG. 3, Wnt3a and aWnt5a-mimetic peptide can stimulate the Fz activity towards Go to asimilar extent as an unrelated known GPCR can stimulate G proteinactivation. These findings are the basis for the assay of the presentinvention to screen for Fz ligands by measuring the non-constitutiveactivity of Fz.

A new and unexpected finding is that Fz ligands can stimulate Fz only athigh concentrations of Go. At these high concentrations the constitutiveactivity of hFz1 is inhibited, but the non-constitutive activity can betriggered. This means that the non-constitutive activity of Fz is alsorestricted to a certain concentration of Go and can be specificallyevoked.

On the basis of the non-constitutive activity of Fz or Fz coupled Gproteins that is restricted to a certain range G protein concentrationthe present inventors developed a new assay for measuring thenon-constitutive activity of Fz. In this assay the concentrations of Gprotein to Fz are provided in ratios, wherein the Fz or the Fz coupled Gproteins are non-constitutively active. The assay makes use of this welldefined restricted concentration range, wherein Fz or Fz coupled Gproteins are non-constitutively active. By experimentally presetting theconcentration ratios of G protein/Fz to high values the assay definesthe kind of activity of Fz that can be triggered by adding ligands.Therefore, the present invention provides an assay for measuringspecifically the non-constitutive activity of Fz.

As mentioned above, Fz show the concentration dependent constitutiveactivity towards G protein. This constitutive activity is sensitive tothe concentration of Gαo-GTP molecules released as a result of thisconstitutive Fz activity, so that the negative feed-back takes place.However, addition of the Fz ligands changes the G protein concentrationdependence of the Fz response. At concentrations of Go optimal forconstitutive Fz activity, addition of Fz agonists decreases thisconstitutive Fz activity. In contrast, at high G protein concentrations,when constitutive Fz activity is low, addition of Fz ligand nowstimulates Fz activity. This stimulation can also be viewed as a reliefof inhibition from high Go concentrations.

In a preferred embodiment of the invention the assay for measuring thenon-constitutive activity and the assay for measuring the constitutiveactivity can be combined consecutively by e.g. changing the provided Gprotein, Fz or membrane protein concentration.

By the assay of the present invention for measuring the non-constitutiveFz activity the influence of Fz ligands e.g. agonists, antagonists,modulators, effectors etc. as well as all kind of modulators andeffectors that are associated with the Fz pathway e.g. effectors thatbind to the G protein can be determined. It can be envisioned that e.g.an antagonist of the non-constitutive Fz activity will have anapplication as a drug reducing Fz activity in e.g. colon cancer ordegenerative disorders.

Therefore, both assays of the invention provides a fast and efficienthigh throughput screening technique to find pharmaceutical modulators ofFz activities that can be preformed by a robot, in a simple, cheap androbust manner with the advantages of a cell free system.

In the most preferred embodiment of the assays of the invention thebinding of GTP to G protein is detected as an indicator for Fz receptoractivation. A further important advantage of this embodiment overcurrently used cell transcription based assays is the possibility ofidentifying substances specifically disrupting interactions ofparticular Fz ligands with particular Fz, rather than substances actingat a common downstream component of the pathway. Therefore, assays areprovided that measure the Fz activity specifically for Fz and/or Fzligands. Additionally, this direct measurement is not reflecting less orno other interfering down- or upstream interactions.

In a preferred embodiment of the invention the assays for measuring aconstitutively or non-constitutively activity of Fz further comprise atleast one frizzled receptor ligand Therefore, the assay is provided in asufficient form.

The assays of the invention can be provided with membrane proteins. Inanother preferred embodiment the assays of the invention can be providedwithout membrane proteins and the membrane proteins or Fz and/or Fzligands can be added afterwards by the user/applicant at the own optionand choice of the user/applicant.

Fz receptor ligands can be added as pure extracts, crude preparations,e.g. homogenized samples or lysis samples or conditioned media etc.and/or can be purified after measuring G protein activation. To purifyFz ligands, protein purification techniques as known in the art areapplied. For example, several chromatography and filtration steps areperformed, whereas eluted fractions are probed for Wnt activity to becollected and applied to further purification.

Outstanding advantages of the assays and methods of the presentinvention are that they are very fast and simple and can be combinedwith other assays and methods. For instance, the medium is applied toseveral chromatography matrixes to purify Wnt ligands from the medium,whereas eluted fractions are probed for Wnt activity to be collected andapplied to further purification. Instead of the currently usedtime-consuming procedures to monitor Wnt activity in these fractions, byapplication of the assays or methods of the present invention thesefractions can be tested for their probability to activate Fz. Thefractions inducing higher than constitutive G protein activation arecollected and used in subsequent purification steps. Therefore, theassays and methods of the present invention provide the possibility toobtain pure and active Fz ligands in high amounts.

The term “frizzled receptor ligand” as used herein refers to all kindsof ligands binding to frizzled receptors. The term “ligand” refers to amolecule that is able to form a complex with a biomolecule e.g. the Fz,G protein to serve a biological purpose. In more specific sense, it isan effector molecule binding to a site on a target protein e.g. Fz, Gproteins, by intermolecular forces such as ionic bonds, hydrogen bonds,and Van der Waals forces.

In a more preferred embodiment the frizzled receptor ligand is selectedfrom the group consisting of at least one agonist, antagonist, inverseagonist, effector, modulator, natural ligand, artificial ligand, andrecombinant ligand.

The term “agonist” as used herein refers to a substance that binds to aspecific receptor and triggers a response in the cell. Also included inthis definition are partial agonists that also bind and activatereceptors, but have only partial efficacy for a receptor compared to afull agonist. They may also be considered as a ligand, which displaysboth agonistic and antagonistic effects. In the presence of fullagonists, partial agonists act as competitive antagonists. Alsocomprised are co-agonists that cooperate with other co-agonists toproduce the desired effect, endogenous, and exogenous agonists. The mostprominent Fz agonists are members of the Wnt protein family.

The term “antagonist” as used herein refers to a ligand that block thebinding of an agonist at a receptor molecule, inhibiting the signalproduced by a receptor-agonist coupling. For example, competitiveantagonists reversibly bind to receptors and compete with other agonistsand antagonists for a specific binding site. Reversible non-competitiveantagonists bind to a different binding-site from the agonists, exertingtheir action to that receptor via the other binding site. They arecalled non-competitive antagonists, because they do not compete for thesame binding site as the agonists. Irreversible antagonists bindcovalently to the receptor binding site. They do not compete withagonists since due to the covalent nature of the bond they can not bedisplaced from the receptor by raising the concentration of an agonist.Examples of Fz antagonists are Dickkopf, WIF (Wnt inhibiting factor) andsFRP (soluble frizzled related protein).

The term “inverse agonist”, also called negative modulator, as usedherein refers to an agent, which binds to the same or different receptorbinding-site as an agonist for that receptor, but exerts the oppositepharmacological effect. Inverse agonists are effective against certaintypes of receptors, which have constitutive activity.

The terms “effector”, also called “modulator”, as used herein refers tomolecules that bind to a protein and thereby alters the activity of thatprotein. A modulator can bind to a regulatory site during allostericmodulation and modulates allosterically the shape of the protein. Apartfrom allosteric modulators, inhibitors and activators are also included.

The term “natural ligand” as used herein refers to physiological ligandsthat are naturally occurring. In contrary, the term “artificial ligand”as used herein refers to artificially synthesized ligands e.g.chemically synthesized ligands. Also comprised are modified ligands.

The term “recombinant ligand” as used herein refers to ligands producedby recombination techniques. Recombinant ligands are encoded byrecombinant, genetically engineered or modified polynucleotides.Recombinant ligands are cloned in vectors and expressed.

In a further preferred embodiment of the invention the frizzled receptorligand and/or frizzled receptor is expressed by a vector, preferably inbacteria, most preferably in Escherichia coli.

The term “vector” as used herein refers to carriers of foreign,recombinant or modified polynucleotides that are cloned into thesevectors for expression, i.e. a vehicle for transferring genetic materialinto a cell. For instance, vectors are bacterial plasmids, viruses likebaculovirus or phages, and yeast vectors. Other examples of preferredbacteria besides Escherichia coli are Shigella flexneri, Salmonellatyphimurium, Bacillus subtilis or Streptomyces coelicolor. Fz can beproduced in mammalian cells e.g. CHO cells to serve as basis for the invitro Fz activation assay. It is obvious that there are many moreapplicable ways to express frizzled receptors, such as production of Fzas inclusion bodies in E. coli with subsequent refolding, baculovirusbased production in Sf9 cells, expression in various yeast strains,insect cell lines, or many other mammalian cell lines, e.g. HeLa, HEK aswell as expression pf Fz in a cell free translation or coupledtranscription translation system of bacterial, wheat germ, rabbitreticulocyte, or other origin.

In another embodiment of the invention the system for measuring Gprotein activity is selected from the group consisting of fluorescent,radioactive and spectrophotometric techniques.

For measuring G protein activity as defined above molecules e.g. GTP orG protein have to be labelled fluorescently, radioactively orspectrophotometrical that their binding can be detected. The radioactivetechnique depends on radioactive labels that are exposed to X-ray filmor measured by scintillation counters. Frequently used radioactiveisotopes in these assays are ¹⁴C, ³²P, ³⁵S and ¹²⁵I. Examples forspectrophotometric detection techniques are fluorescent and colorimetricmethods with spectral properties in different ranges of wavelengths. Thefluorescent detection technique depends on fluorescent labels likeEuropium, Oregon Green, Texas Red, or GFP that are excited by properkinds of light, and the emission of the excitation is then detected by aphotosensor, such as CCD camera equipped with appropriate emissionfilters. The colorimetric detection technique depends on labelling bydyes with different colours that absorb or emit in the range of visiblelight, whose amount or concentration is measured via densitometry orspectrophotometry. Also comprised are chemiluminescent detection methodsthat depend on luminescent molecules like enhanced chemiluminescence.

A further aspect concerning the present invention is a method formeasuring a constitutive or non-constitutive activity of a frizzledreceptor comprising the steps of:

-   -   a) providing at least one membrane protein with at least one        cell free frizzled receptor;    -   b) adding at least one frizzled receptor ligand and at least one        G protein; and    -   c) incubating the cell free frizzled receptor, the frizzled        receptor ligand and the G protein in a system for measuring G        protein activity, wherein the detection of guanosine        triphosphate (GTP) binding to the G protein indicates the        activity of the G protein.

To measure a constitutive or non-constitutive activity of a frizzledreceptor at least one cell free frizzled receptor, at least one frizzledreceptor ligand and at least one G protein, which have been defined bythe above explanations are combined in one tube or well of a plate. Inaddition, GTP analogue nucleotide such as Europium-GTP, optionallyguanosine diphosphate (GDP) and the required buffers and salts areprovided. These components were incubated in a system for measuring Gprotein activation that is selected from the group consisting offluorescent, radioactive and colorimetric techniques. G proteins can becrude preparations, purified or non-purified extracts from naturalsources or artificially produced.

The method for measuring constitutive or non-constitutive activity of Fzis based on the GDP/GTP exchange by activated G proteins. Thereforethere is a variety of parameters that can be used directly or indirectlyfor monitoring the GTP binding to the G protein. For instance, thebinding of GTP itself, the uncoupling of GDP by detecting the amount ofmembrane bound GTP or GDP, respectively, and the amount of free GTP orGDP can be detected.

One system to monitor G protein activation is based on the Europiumlabelled GTP analogue and time-resolved fluorescence measurements. Otherpotential systems are e.g. the classical filter-based ³⁵S-GTPγS assay orthe fluorescence enhancement assays using BODIPY-GTPγS molecules thatenhance their fluorescence upon binding to G proteins.

In preferred embodiments Fz receptor ligands used for the method of theinvention are expressed by a vector preferably in bacteria, mostpreferably in Escherichia coli, and are selected from the groupconsisting of at least one agonist, antagonist, inverse agonist,effector, modulator, natural, artificial and recombinant ligand as aforementioned.

A further aspect of the invention concerns a method for obtaining anactive non-mammalian frizzled receptor ligand comprising the steps of:

-   -   a) performing random and/or directed mutagenesis in at least one        polynucleotide sequence encoding at least one inactive frizzled        receptor ligand to generate a mutated polynucleotide sequence;    -   b) digesting the mutated polynucleotide sequence into random        polynucleotide fragments;    -   c) recombining the random polynucleotide fragments to obtain        recombinant polynucleotides by at least one recombination        technique;    -   d) cloning the recombinant polynucleotide into a vector;    -   e) expressing the recombinant polynucleotide resulting in        recombined frizzled receptor ligands;    -   f) measuring the ability of the recombinant frizzled receptor        ligands to activate frizzled receptors in a system comprising at        least one G protein and at least one membrane protein with at        least one frizzled receptor; and    -   g) repeating step a) to f) until the recombinant frizzled        receptor ligands activate the G protein at least 10-fold,        preferably 100-fold, more preferably 500-fold, most preferably        1000-fold compared to frizzled receptor ligands encoded by the        polynucleotide sequence of step a).

The term “polynucleotide” as used herein refers to chain moleculescomprising more than about 10 nucleotides.

The term “active frizzled receptor ligand” as used herein refers to Fzligands that are able to activate Fz in general. The term “inactivefrizzled receptor ligand” as used herein refers to completely inactiveand less active Fz ligands that are only able to activate Fz to a lowextent.

The term “cloning” as used herein refers to molecular cloning, i.e. aprocess to create identical copies of polynucleotides. The term “randommutagenesis” as used herein refers to random mutagenesis methods thatintroduce changes at positions throughout the polynucleotide sequence.In most of these techniques copying of a DNA sequence is deliberatelydisturbed. These methods include the use of physical and chemicalmutagens, mutation strains and some forms of insertion and deletionmutagenesis as well as the various forms of error-prone PCR. Randommutations result from improper polynucleotide replication or inadequaterepair of DNA damage. The term “directed mutagenesis” as used hereinrefers to directed mutagenesis methods that randomize only a specificposition within a polynucleotide sequence. These methods involve thedirect synthesis of mixtures of DNA molecules and are usually based onthe incorporation of partially randomised synthetic DNA cassettes intogenes via PCR or direct cloning. The key to these methods is theintroduction of diversity at specific positions within the syntheticDNA. The term “recombination technique” as used herein refers tomethods, such as DNA shuffling and staggered extension process that takeportions of existing sequences and mix them in novel combinations. Thesemethods do not directly create new sequence diversity, but combineexisting diversity in new ways. Recombination techniques comprisinghomologous and non-homologous methods make it possible to bring togetheradvantageous mutations while removing deleterious mutations in a manneranalogous to sexual recombination. Methods such as iterative truncationfor the construction of hybrid enzymes that make it possible toconstruct hybrids proteins even when the genes have little or nosequence homology also belong to this category. Directed, randommutagenesis and recombination techniques are summarized by the genericterm “protein evolution techniques”.

By the method of the present invention for obtaining an active frizzledreceptor ligand it is possible to evolve Wnt ligands for efficientproduction in non-mammalian hosts, ideally Escherichia coli. Currently,bacterially-produced Wnt ligands lack biological activity. It ispossible to apply protein evolution techniques, such as proteinshuffling, to evolve Wnt ligands to a new form with improve biologicalactivity, foldability and stability upon production in bacteria.

The term “non-mammalian frizzled receptor ligand” as used herein refersto Fz ligands that are produced in organisms that are not members of theclass mammalian, e.g. in prokaryotic, insect, yeast cells or by viruses.Also comprised are Fz ligands produced by vectors or expression systems.

The principle of the method of the present invention is a combination ofa mutagenesis technique e.g. error-prone PCR amplification of theinitial sequence with subsequent recombination techniques by digestioninto random-size fragments followed by re-assembly, repeated severalcycles. In other words, this is a combination of random incorporation ofnucleotide changes with recombination. At the end of each cycle, thelibrary of resulting variants of the initial sequence is cloned into avector, transformed into a host e.g. Escherichia coli, expressed, andprobed for activity. If the activity of a clone is increased, it isisolated for the next cycle of protein evolution until the activity ismultiplied. This technique can be used to improve protein activitiestens of thousand times. Therefore, the method of the present inventionprovides the possibility to obtain pure and active Fz ligands in highamounts.

The existence of an easy and fast assay to monitor Wnt activity isessential for such a method. In a preferred embodiment of the inventionthe assay for measuring constitutive or non-constitutive activity of Fzof the present invention is used for this purpose.

In a preferred embodiment of the methods according to the invention aconcentration of the G protein is approximately 0.4 μg/ml or less,preferably between approximately 0.002 μg/ml and 0.3 μg/ml, morepreferably between approximately 0.005 μg/ml and 0.15 μg/ml, mostpreferably approximately 0.1 μg/ml in relation to approximately 2 μg/mlto 15 μg/ml of the membrane protein.

In another preferred embodiment of the methods according to theinvention a concentration of the G protein is approximately 0.3 μg/ml orhigher, preferably between approximately 0.035 μg/ml and approximately10 μg/ml, more preferably approximately 0.4 μg/ml, most preferablyapproximately 0.5 μg/ml in relation to approximately 2 μg/ml to 15 μg/mlof the membrane protein.

In a preferred embodiment of the invention the frizzled receptor ligandis a Wnt protein. The term “Wnt” as used herein refers to a huge familyof signalling molecules of the Fz pathway. These proteins contain asignal sequence of 350-380 amino acids followed by a highly conserveddistribution of cysteines. Although Wnt proteins are secreted, they showan insoluble nature that has been explained by the discovery that theseproteins are palmitoylated and are more hydrophobic than initiallypredicted from the primary amino acid sequence. Until now theinsolubility of Wnt has impeded methods for purifying Wnt and precludedan isolation of Wnts in high quantities. However, the assays and methodsof the present invention overcome this problem and provide thepossibility to yield pure and active Wnt in high amounts.

In a preferred embodiment of the invention wherein the step of providingat least one cell free frizzled receptor and/or the step of adding atleast one frizzled recepfor ligand and at least one G protein is/arepreceded by at least one protein evolution technique, as describedabove.

A further aspect of the invention is the use of the assays of thepresent invention for screening frizzled receptor ligands and/or formeasuring levels of frizzled receptor ligands and/or for measuringlevels of frizzled receptors and/or for obtaining active frizzledreceptor ligands. The term “levels” as used herein refers to amounts,volumes, concentrations and ratios. Obtaining active Fz ligand in highamounts by a cheap and easy way is great benefit, because ligands can beused e.g. as growth factors, promoting the maintenance and proliferationof stem cells.

A further aspect of the invention is the use of the assays of thepresent invention, wherein the frizzled receptor ligands are drugcandidates, preferably drugs for cancer diseases and degenerativedisorders. Evidently, there is a broad variety of diseases that can betreated with pharmaceuticals influencing Fz pathways.

Mis-regulation of the Wnt pathway leads to a variety of abnormalitiesand degenerative diseases, like tetra-amelia, bone density defects,vascular and retinal defects in the eye, tooth agenesis, colorectal andcolon cancer. Mutations that promote constitutive activation of the Wntsignaling pathway lead to cancer. In addition to tooth defects,individuals with Axin2 mutations display for instance a prediposition tocolon cancer. Moreover, alterations of Wnts, APC, axin, and TCFs are allassociated with carcinogenesis. The best-known example of a diseaseinvolving an overactivation of Wnt pathway that produces tumors isfamilial adenomatous polyposis (FAP), an autosomal, dominantly inheriteddisease, in which patients display hundreds or thousands of polyps inthe colon and rectum. Aberrant activation of theWnt/Frizzled/beta-catenin signaling pathway leads to tumorigenesis inmany tissues. The majority of breast cancers are associated withstabilization of beta-catenin, which is a hallmark of overactivation ofthis pathway. Specifically, Wnt2, Wnt5a, Wnt7b, Wnt10b, Wnt13/2b, andWnt14 have been reported as overexpressed in breast cancer as comparedto normal breast tissue. Further, most breast cancer cell linesoverexpress several Wnt ligands including Wnt3a, Wnt4, Wnt6, Wnt8b,Wnt9a, and Wnt10b. In contrast, expression of the secreted Wntinhibitors sFRP1 and WIF1 is reduced or lost in most cases of breastcancer. Thus, antagonists of Wnt/Frizzled interactions and moleculesinhibiting Fz pathway become immediate anticancer drug candidates. Inaddition, it gets obvious that Wnt overactivation is a reason for agingand decreased synapse formation. Therefore, inhibitors of Fz pathway canbe antidegenerative and memory enhancing drugs.

The embodiments described for the assay of the invention can also beapplied to the methods of the invention and vice versa.

DETAILED DESCRIPTION OF THE FIGURES FIG. 1. Recombinant Human FrizzledReceptor 1 (hFz1) can be Produced In E. Coli Membranes and canPhysically Bind the Heterotrimeric G Protein Go

(A) Membrane fractions of control bacteria and bacteria expressing theMBP (maltose binding protein)-hFz1 fusion protein. Two bacterial cloneswere used, which differ in the level of MBP-hFz1 expression. Westernblotting with antibodies to MBP was done. Multiple bands representdifferent states of the receptor unfolded by sodium dodecyl sulphate(SDS).

(B) hFz1 membranes, but not control bacterial membranes, bind Go.Membranes from (A) were incubated with bovine brain Go heterotrimeric Gprotein, followed by ultracentrifugation. Amounts of Go in thesupernatant (S) and pellet (P) were compared. While control membranesfail to bring Go to the pellet, hFz1 membranes co-precipitate Go.Importantly, hFz1 membranes from clone 2 which express higher levels ofhFz1 (see (A)) bind more Go, demonstrating the direct hFz1-Gointeractions.

FIG. 2. hFz1 Showed a Basal G Protein-Activating Activity

hFz1 was expressed in CHO cells by means of transient transfection.Plasma membranes from these cells, as well as from controlnon-transfected CHO cells of a similar density, were prepared asdescribed in Materials and Methods. G protein-activating activity of thehFz1 membranes was compared to that of control membranes using thePerkin Elmer Europium-GTP TRF assay at varying amounts of theectopically added bovine brain Go protein. At proper membrane/Go ratios,hFz1 demonstrates about 3-fold increase of Go protein activation overcontrol membranes.

FIG. 3. Wnt Ligands can Further Stimulate hFz1 Activity Towards Go

(A) CHO-produced hFz1 membranes were stimulated with 5 μlWnt3a-conditioned medium per 100 μl of the reaction volume. E.coli-produced hFZ1 membranes were stimulated with 0.3 μg/ml purifiedWnt3a. For comparison, CHO membranes expressing human fMLP receptorhFRP1 as an example of an unrelated known GPCR from Perkin Elmer werestimulated to a similar extent with 1 μM formyl-peptide ligand.

(B) E. coli produced hFz1 membranes can be stimulated with the Wnt5amimetic peptide fMDGCEL (Säfholm A. et al, 2006).

FIG. 4. Detergent-Solubilized hFz1 Stimulated GTP Incorporation into Goin the Presence of Different Wnt Ligands

Addition of Wnt5a, Wnt5b, Wnt7a induced concentration-dependentactivation of the trimeric Go protein in the presence of hFz1-expressingmembrane fractions, but not control membranes. Among the Wnt ligandstested, Wnt3a and Wnt5a were most active, while Wnt5b and Wnt7ademonstrated a more modest stimulation (FIG. 4). Statisticalsignificance is shown as “*”, “**”, and “***” (P value by unpairedt-test <0.05, <0.005, and <0.001, respectively).

FIG. 5. Wnt Ligands Stimulate Several Frizzled Receptors to Activate theTrimeric G Proteins

To demonstrate that other Frizzled receptors than hFz1 can beinvestigated by the in vitro G protein activation assay according to theinvention, human Frizzled receptors 6 and 7 (hFz6, hFz7) werebacterially expressed. When tested for their ability to activate Go uponaddition of different ligands, hFz6 activated Go in the presence ofWnt7a and to a lesser extent Wnt5a (FIG. 5A), while hFz7 was mostlystimulated by Wnt5a (FIG. 5B). The results obtained with three humanFrizzled receptors (hFz1, hFz6, hFz7) and four Wnt ligands (Wnt3a,Wnt5a, Wnt5b, and Wnt7a) allow to compose a table of pair-wiseWnt-Frizzled activations using Go stimulation as the read-out (FIG. 5C).

Detergent-solubilized hFz6 and Fz7 stimulated GTP incorporation into Goin the presence of different Wnt ligands. Data are presented in FIG. 5Cas summary of the capacity of the tested Wnt ligands to stimulate thetested Frizzled receptors to activate Go in vitro. Control membranesshowed no Go activation in the presence of any Wnt. Go activation by theWnt-Frizzled pairs is denoted (in the order of the decreasing activationcapacity) as “+++”, “++”, “+”, “+/−”, and “−” using as criteria thelowest Go-activating Wnt concentration as well as the maximal Goactivation level achieved by any Wnt concentration.

Materials and Methods 1. HFz1 Expression in Bacteria

The coding sequence of hFz1 was cloned in-frame into the pMAL-p2 plasmid(New England BioLabs) allowing expression of the protein as anN-terminal fusion with maltose binding protein (MBP). The natural signalsequence of MBP directs the fusion protein through the cytoplasmicmembrane. Expression conditions were optimized to allow slow expressionto maximize correct hFz1 folding: Expression was performed at 18° C. for18 h, and in the presence of glucose to minimize the expression ofendogenous MBP protein.

Bacteria were harvested by centrifugation, resuspended in PBS(phosphate-buffered saline) and disrupted by French press. The resultantsuspension was cleared from the cell debris by low-speed centrifugation,followed by ultracentrifugation to pellet the membranes expressingMBP-hFz1. The membranes were resuspended in PBS supplemented with theprotease-inhibitor cocktail (Roche) and frozen in aliquots at −80° C.Control bacteria were transformed with the empty pMAL-p2 vector, inducedwith IPTG, grown and harvested in parallel with hFz1-transformedbacteria.

2. HFz1 Expression in Cho (Chinese Hamster Ovary) Cells.

HFz1 coding sequence was subcloned into pIRES2-DsRed-Express plasmid(Invitrogen). CHO cells were transiently transfected with lipofectaminefollowing manufacturer's protocol. Transfection efficiency was estimatedto be >50%. Two days post-transfection, transfected cells in parallelwith control non-transfected cells of a similar cell density werewashed, detached with EDTA, pelleted, washed with PBS, and disrupted ina glass-rod homogenizer in a hypotonic buffer (10 mM Hepes pH 7.5).Membrane fraction was prepared and stored as above.

3. Europium-GTP Assay of G Protein Activation

Europium-GTP assay of G protein activation using a time-resolvedfluorescence assay was performed. In one well of a 96-well AcroWellplate, 100 μl were mixed containing 4-10 μg/ml membrane protein from thehFz1 expression in bacteria or in CHO, 50 mM Hepes, pH 7.4, 1 mM MgCl₂,50 mM NaCl and 1 mg/ml saponin. Bacterial membranes expressing hFz1 orbacterial control membranes were used at 0.1 mg/ml protein concentrationfor membrane protein preparation. CHO membranes expressing hFz1 or CHOcontrol membranes were used at 0.01 mg/ml protein concentration. Varyingamounts of the following were added: bovine brain Go (Calbiochem), Wnt3a(R&Dsystems), Wnt3a-conditioned or control L-cell medium. Extra GDP wasomitted in most experiments. 30 min after incubating this mixture atroom temperature on a shaker (100 rpm), Europium-GTP was added, andincubation was continued for 30 more min. Afterwards the content of thewells was filtered and washed using the Multiscreen Vacuum Manifold(Millipore). Amounts of Europium-GTP retained by the membranes were thenmeasured using the Perkin Elmer Victor3 Multilabel Plate Reader.

4. DNA Shuffling as a Directed Protein Evolution Technique

Point mutations are induced by error-prone PCR into wnt genes generatinga library of Wnt coding DNA with randomized point mutations. Therefore,Mn²⁺ or Mg²⁺ is added to a standard PCR reaction mixture with wnt genesto impose imperfect reaction conditions.

This library was the substrate for the subsequent DNA shufflingreaction. For DNA shuffling, mutated DNA is digested into randomfragments with DNase I. Fragments are resuspended in a standard PCR mixwith different synthetic chimeric oligonucleotides for amplification,wherein the fragments are spliced together randomly. The spliced wntgene fragments are then assembled by primerless PCR. Individualfragments prime against each other to recreate a full-length recombinantwnt gene. Diversity in this DNA shuffling was controlled by the numberof different mutated wnt genes recombinant. The resulting new genelibrary is cloned into bacterial vector pMAL-p2, transformed andexpressed for testing the ability of Wnt to activate G protein.

5. Wnt Protein Purification

To purify Wnt protein as an example for a typical Fz ligand LWnt3A orLWnt5A cell lines producing and secreting Wnt proteins were used. Bygrowing the cells adherent for 4 days from a 1:10 to 1:20 split highamounts of Wnt-3A were obtained. The medium was DMEM plus 10% FBS,omitting the serum lowers the levels of Wnt protein in the medium. TheWnt containing medium was filtered, detergent was added (Triton X-100 orCHAPS) to a final concentration of 1%, and the medium was refilteredjust before applying the material to Blue Sepharose.

Purification by Blue Sepharose fractionation yielded pure Wnt protein,wherein Wnt3A conditioned medium was applied on a Blue Sepharose HPcolumn. Wnt elution from Blue Sepharose was done in a single step from150 mM to 1.5 M KCl. Approximately, half of the Wnt protein elutedimmediately with the salt and the majority of contaminants, but theother half was retained and eluted later in a second pool. This secondpool contained much less total protein and consequently contained ahigher proportion of Wnt.

Pooled eluted Wnt fractions from Blue Sepharose purification wereconcentrated by gel filtration to 5 or 10 ml volume using Centricon orAmicon Ultra 30 ultrafiltration device (Amicon) depending on size of gelfiltration column.

A heparin cation exchange chromatography served the purpose of furtherconcentrating the protein and removing predominant contaminants as mostlikely BSA. The final concentration of Wnt3A was about 0.1 mg/ml. If theconcentration is higher, a precipitate will form which is predominatelyWnt protein. This precipitate can be pelleted easily. The remainingsupernatant contains Wnt3A at about 0.1 mg/ml while the pellet containslargely inactive Wnt protein. Therefore, the present conditions (1X PBS,1M NaCl, 1% CHAPS, pH7.3) could maintain the solubility and activity ofWnt3A at a maximum of 0.1 mg/ml.

All purification steps were carried out at 4° C., and purified proteinwas also stored at 4° C. Protein can also be freeze-dried with retentionof activity.

REFERENCES

-   Borchert K. et al., Assay and Drug Development Technologies, 3(2),    133-141, 2005.-   Brennan K. and Brown M., Journal of Mammary Gland Biology and    Neoplasia, 9(2), 119-131, 2004.-   DasGupta R. et al., Science 308, 826, 2005.-   Huang H. and Klein P., Genome Biology, 5 (234), 1-7, 2004.-   Le Garrec J. et al., Developmental Dynamics, 235, 235-46, 2006-   Logan C. and Nusse R., Annu. Rev. Cell Dev. Biol., 20,781-810, 2004.-   Säfholm A. et al., J. Biol. Chem., 281(5), 2740-9, 2006.-   Willert K. et al., Nature 423, 448-452, 2003.

1-6. (canceled)
 7. A method for measuring a constitutive activity of afrizzled receptor comprising the steps of: a) providing at least onemembrane protein with at least one cell free frizzled receptor; b)adding at least one frizzled receptor ligand and at least one G protein;and c) incubating the cell free frizzled receptor, the frizzled receptorligand and the G protein in a system for measuring G protein activity,wherein the detection of guanosine triphosphate (GTP) binding to the Gprotein indicates the activity of the G protein.
 8. The method of claim7, wherein a concentration of the G protein is approximately 0.4 μg/mlor less.
 9. (canceled)
 10. The method of claim 7, wherein the frizzledreceptor ligand is a Wnt protein.
 11. The method of claim 7, whereinstep a) and/or step b) are/is preceded by at least one protein evolutiontechnique. 12-13. (canceled)
 14. The method of claim 7, wherein thefrizzled receptor ligands are drug candidates.
 15. The method of claim14, wherein the drug candidates are for cancer.
 16. The method of claim14, wherein the drug candidates are for degenerative disorders selectedfrom the group consisting of tetra-amelia, bone density defects,vascular and retinal defects in the eye, and tooth agenesis.
 17. Amethod for measuring a non-constitutive activity of a frizzled receptorcomprising the steps of: a) providing at least one membrane protein withat least one cell-free frizzled receptor; b) adding at least onefrizzled receptor ligand and at least one G protein; and c) incubatingthe cell-free frizzled receptor, the frizzled receptor ligand and the Gprotein in a system for measuring G protein activity, wherein thedetection of guanosine triphosphate (GTP) binding to the G proteinindicates the activity of the G protein.
 18. The method of claim 17,wherein a concentration of the G protein is approximately 0.3 μg/ml orhigher.
 19. The method of claim 17, wherein the frizzled receptor ligandis a Wnt protein.
 20. The method of claim 17, wherein step a) and/orstep b) are/is preceded by at least one protein evolution technique. 21.The method of claim 17, wherein the frizzled receptor ligands are drugcandidates.
 22. The method of claim 21, wherein the drug candidates arefor cancer.
 23. The method of claim 21, wherein the drug candidates arefor degenerative disorders selected from the group consisting oftetra-amelia, bone density defects, vascular and retinal defects in theeye, and tooth agenesis.